The green fluorescent protein (GFP) from Aequorea victoria was discovered in 1962. Knowledge of the structure, mechanism and applications of GFP developed very rapidly after cloning of the GFP gene in 1992. After demonstration of the heterologous expression of the GFP gene in other organisms, GFP became one of the most widely used reporter genes. In 1999, another family of fluorescent proteins, including Discosoma Red (DsRed), was cloned from corals followed by Anemonia asRed in 2000 and HcRed in 2001. The biological role of these proteins extends from a pure signal function as in GFP to photoprotection of photosynthetic symbionts by the novel proteins isolated from corals. For two of these colored, water-soluble proteins, GFP and DsRed, with a molecular weight of 25-27 kD, X-ray structure analysis has demonstrated a homologous β-barrel structure. A common feature of the primary structure of these proteins is that the amino acids, tyrosine and glycine, which occupy GFP positions 66 and 67, are conserved and participate in the formation of the chromophore during a post-translational autocatalytic modification.
These proteins (wild type and mutants) can be used as multicolor reporters. The spectral range of their fluorescence spans almost 180 nm extending from the “blue” peak position of 460 nm to 640 nm in the red region of the spectrum. GFP is one of the most widely-used genetic markers in cell biology (Gerdes, H.-H., and Kaether, C., FEBS Letters (1996) 389:44-47), in immunology (Kawakami, N., et al., Immunology Lett (1999) 70:165-171), as well as in studies of infectious disease, e.g.—of host-pathogen interaction on model animals (Zhao, M., et al., Proc. Natl. Acad. Sci. (2001) 98:9814-9818). GFP has been used for whole-body imaging of tumor growth, metastasis, and angiogenesis (Yang, M., et al., Proc. Natl. Acad. Sci. (2002) 99:3824-3829; Yang, M., et al., Proc. Natl. Acad. Sci. USA (2000) 97:1206-1211; and Yang, M., et al., Proc. Natl. Acad. Sci. USA (2001) 98:2616-2621), gene expression (Yang, M., et al., Proc. Natl. Acad. Sci. USA (2000) 97:12278-12282), and bacteria infection (Zhao, M., et al, supra).
In all these applications, the red emission is of special importance with respect of minimization of background emission and in vivo scattering as well as for FRET (fluorescence resonance energy transfer) analysis. High extinction coefficients, quantum yield, and the monomeric state of fluorescent proteins are very important parameters for their use as reporters including in vivo applications. In contrast to GFP, which has only a small tendency to dimerize, the related proteins have a pronounced tendency to form oligomers, e.g.—tetramers as observed for DsRed, or even higher aggregates. Extinction coefficients and quantum yields are also relatively low for red proteins and for the newly developed monomeric DsRed.
The human histone H2B gene has been fused to the gene encoding the GFP of Aequorea victoria and transfected into human cells to generate stable lines constitutively expressing H2B-GFP. The H2B-GFP fusion protein was incorporated into nucleosomes without affecting cell cycle progression. H2B-GFP allowed high-resolution imaging of nuclei including both mitotic chromosomes and interphase chromatin, and the latter revealed various chromatin condensation states in live cells (Kanda, T., et al., Curr. Biol. (1998) 8:377-385).
The disclosures of the cited documents are incorporated herein by reference.
These various proteins have been used to monitor tumor metastases, and as reporter genes to monitor expression. See, for example, Kanda, T., et al., Curr. Biol. (1998) 8:377-385; Yang, M, et al., Clin. Exp. Metastasis (1999) 17:417-422; Yang, M, et al., Proc. Natl. Acad. Sci. USA (2001) 98:2616-2621; and Yang, M, et al., Proc. Natl. Acad. Sci. USA (2002) 99:3824-3829. However, to applicants' knowledge, these proteins have not been used to monitor cellular proliferation, cell cycle status, or drug sensitivity of cells in vitro, nor have dual labeled cells been used either in vitro or in vivo.
Most in vitro techniques for monitoring proliferation involve observation techniques which results in killing the cells. For example, cells that proliferate attached to a plastic surface may be released from the plastic by enzymes, such as trypsin, and then counted using a particle counter. Also commonly employed are stains such as tetrazolium dyes which are reduced by electrons derived from mitochondrial enzyme activity and negatively affect the viability of the cells. In addition, the lacZ gene may be introduced into the cells and used as a marker, but in order to visualize activity, the cells must be stained.
Thus, traditional methods of monitoring proliferation involve disruption of normal cellular metabolism, even resulting in cell death.
The present invention offers an opportunity to observe proliferation and other cellular activity in vitro or in vivo in real time. These observations may also extend to observing the cell cycle by taking advantage of changes in nuclear/cytoplastic ratios at various stages.
To applicants knowledge, although the H2B gene fused to GFP has been used to label the nucleus, no suggestion has been made to separately label the nucleus and cytoplasm of living cells with two different color proteins. The present invention provides the capability to observe such living cells.